Method and composition for treating immune complex associated disorders

ABSTRACT

The present invention provides methods and compositions for treating immune complex associated diseases (ICAD), such as SLE, rheumatoid arthritis, and hepatitis-C related immune complex disease (e.g., cryoglobulinemia) in a subject having an ICAD or at risk for developing ICAD. The invention is based upon the surprising finding that chromatin-containing immune complexes activate autoreactive B cells and dendritic cells by a dual receptor engagement process which, in both cell types, involves a Toll-like receptor (TLR). The methods of treating ICAD comprise administering a compound to an individual in need thereof that either 1) inhibits formation of the immune complex either by preventing formation and/or binding to the TLR, or 2) interferes with binding of an autoantigen-containing immune complex (or the antigenic component thereof) to the TLR, or 3) inhibits signaling pathways initiated by dual engagement of BCR and TLR (in B cells) or FcR and TLR (in dendritic cells) via immune complexed or uncomplexed autoantigens.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/487,885, having a 371(c) date of Sep. 27, 2004, which is a nationalstage of PCT/US02/28708, filed Sep. 9, 2002, which claims benefit ofU.S. Patent Application No. 60/367,578, filed Mar. 26, 2002, and U.S.Patent Application No. 60/318,096, filed Sep. 7, 2001.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government Support under Contract Nos. RO1AR-35230 and K08 DK-02597 awarded by the National Institutes of Health.The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and compositions for treatingimmune complex associated diseases, preferably systemic lupuserythematosus (SLE), and other systemic autoimmune diseases associatedwith the subject having aberrant Toll-like receptor (TLR)/B cellreceptor (BCR) dual engagement (in B cells) or TLR/Fc gamma receptordual engagement (in dendritic cells and/or macrophages).

2. Background

Autoimmune diseases are a fairly common but poorly understood group ofdiseases in which an individual's immune system either 1) beginsrecognizing self antigens as foreign and starts destroying tissuesexpressing such antigens thereby causing a disease, or 2) forms immunecomplexes with these antigens which then deposit in tissues and causeinflammatory pathology. Such autoimmune diseases include, for example,diabetes wherein the immune system turns against and destroys insulinproducing pancreatic islet cells; multiple sclerosis, wherein the targetantigen is the myelin sheath protecting neurons leading to destructionof function of motor neurons; psoriasis, where the target of the immunesystem is skin; rheumatoid arthritis, where the target organ iscartilage; and systemic lupus erythematosus (SLE), which presents itselfas targeting a variety of tissues with no apparent specificity orselectivity although the target antigens themselves are extremelyconsistent and characteristic. Because the mechanisms leading to thedevelopment of autoimmune diseases in general are mostly unknown, theirtreatment is often directed to generally suppressing the immune system.Such general immunosuppressive therapies often cause a variety ofundesirable side effects including cancer, infertility, and increasedsusceptibility to infections by viruses, fungi, yeast, and bacteria.Therefore, it would be desirable to understand the mechanisms that causethe immune system to turn against self antigens to enable development ofmore specific therapies for the treatment of the autoimmune diseases.

An example of a poorly understood autoimmune disease is systemic lupuserythematosus (SLE), commonly known as Lupus. SLE is characterized bydysregulation of the immune system resulting in the production ofantinuclear antibodies, the generation of circulating immune complexes,and the activation of the complement system. The immune complexes buildup in the tissues and joints causing inflammation, and degradation toboth joints and tissues. While the word “systemic” correctly suggeststhat the disease affects the entire body and most organ systems, thedisease most often involves inflammation and consequent injury to thejoints, skin, kidney, brain, the membranes in body cavities, lung,heart, and gastrointestinal tract. An individual with SLE oftenexperiences unpredictable acute episodes or “outbreaks” and equallyunexpected remissions. The pathologic hallmark of the disease isrecurrent, widespread, and diverse vascular lesions resembling a rash orchanges on the surface of the skin.

The prevalence of SLE in the United States is an issue of some debate.Estimates of prevalence range from 250,000 to 2,000,000 persons.Although reported in both the extremely old and the extremely young, thedisease mainly affects women of childbearing age. Among children SLE isthree times more common in females than in males. In the 60% of SLEpatients who experience the onset of this disease between puberty andthe fourth decade of life, the female to male ratio is 9:1. Thereafter,the female preponderance again falls to that observed in prepubescentchildren (i.e. 3:1). In addition, the disorder appears to be three timesmore common in persons of African and Asian descent than in persons ofCaucasian descent.

The etiology of SLE remains unknown. A genetic predisposition, thesystemic proliferation of sex hormones, and various environmentaltriggers, such as viral infections have been suggested to play a role intriggering the aberrant immune responses that typify the disease. A rolefor genetics is suggested by the increased percentage of twohistocompatibility antigens, HLA-DR2 and HLA-DR3, in patients with SLE.In addition, there is an increased frequency of the extended haplotypesHLA-A1, B8, and DR3 in affected individuals. The role of heredity isfurther supported by the concordance for this illness among monozygotictwins. The polygenic nature, however, of this genetic predisposition aswell as the contribution of environmental factors is suggested by theconcordance rate, which is only moderate and reported to be between 25%and 60%.

The precise initiating etiology of SLE is unknown. However, it isgenerally accepted that most of the clinical manifestations of thedisease are caused either directly or indirectly by autoantibodyproduction and the subsequent formation of pathogenic immune complexes.These autoantibodies, which are produced by dysregulated B lymphocytes,have distinct specificities recognizing discrete nuclear autoantigensincluding, among others, DNA, nucleosomes and subnucleosomes. CertainRNA/protein complexes including the Sm antigen and small nuclearribonucleoproteins (snRNP) are additional characteristic autoantigenicspecificities. The pathogenic immune complexes are formed by binding ofthe autoantibodies to their respective nuclear autoantigens.

Autoantibodies in SLE often circulate as immune complexes (IC) boundwith their respective autoantigens. Chromatin or chromatin fragmentssuch as DNA, nucleosomes or subnucleosome particles are especiallycommon autoantigenic specificities in both mice and humans (Tan, E. Adv.Immunol. 44, 93-151 (1989); Monestier and Novick, Mol. Immunol. 33:89-99., 1996)

The central goals in the treatment of SLE, therefore, are either toattempt to suppress the dysfunctional B lymphocytes thereby decreasingthe production of autoantibody or, to attempt to diminish thepathogenicity of the immune complexes once they have formed. At presentthese goals can only be achieved, and often incompletely so, by the useof intensive systemic immunosuppressive drug therapy using drugs such ascortisone, azathioprine, hydroxychloroquine and cyclophosphamide. Thesetherapies are associated with many serious and undesirable side-effectsincluding infections, infertility, retinopathy and cancer. Therefore,new treatments for SLE, and other autoimmune diseases, would bedesirable.

SUMMARY OF THE INVENTION

It is therefore the purpose of the present invention to provide methodsand compositions for treating immune complex associated diseases (ICAD),such as SLE, rheumatoid arthritis, and hepatitis-C related immunecomplex disease (e.g., cryoglobulinemia) in a subject having an ICAD orat risk for developing ICAD.

We have discovered that chromatin-containing immune complexes activateautoreactive B cells and dendritic cells by a dual receptor engagementprocess. In both cell types a Toll-like receptor (TLR) is involved.TLR9, located in a cytoplasmic compartment, is the essential secondreceptor required for cell activation. In the case of the B cell, the Bcell antigen receptor located on the cell surface is the essential firstreceptor required for cell activation. In the case of the dendriticcell, a stimulatory Fc gamma receptor located on the cell surface is theessential first receptor required for cell activation. We have found amethod of treating ICAD by administering a compound to an individual inneed thereof that either 1) inhibits formation of the immune complex(i.e., autoantibody and nuclear autoantigen) either by preventingformation and/or binding to the Toll-like receptor (TLR), or 2)interferes with binding of an autoantigen-containing immune complex (orthe antigenic component thereof) to the TLR, or 3) inhibits signalingpathways initiated by dual engagement of BCR and TLR (in B cells) or FcRand TLR (in dendritic cells) via immune complexed or uncomplexedautoantigens. The compound is administered in a pharmaceuticallyacceptable carrier.

Preferably, the ICAD is SLE, rheumatoid arthritis or hepatitis-C relatedimmune complex disease (e.g., cryoglobulinemia). In an other embodiment,the ICAD is related to an immune reaction in a host after organtransplantation.

Although not working to be bound by theory, we believe that immunecomplexes (IC) containing an autoantigen, such as chromatin, but not ICcontaining a foreign antigen, are able to activate autoreactive B cellsand that this activation is absolutely dependent on the ability of theautoantigen-containing IC to sequentially engage either the B cellreceptor (BCR) in B cells or FcγR in dendritic cells, and a secondreceptor, a Toll-like receptor. This finding establishes a novel linkbetween the innate and adaptive immune systems and suggests a generalmechanism whereby autoreactive B cells or dendritic cells specific forprotein/nucleic acid autoantigens are activated.

According to one aspect of the invention a method is provided fortreating a patient having an ICAD or at risk for an ICAD by identifyingan individual with ICAD and administering an effective amount of acompound capable of inhibiting the autoantigen or autoantigen/immunecomplex from forming and/or from activating B cells or dendritic cells.

A person at risk for ICAD or systemic autoimmune disease includesindividuals having at least a parent, grandparent or sibling who has anICAD or systemic autoimmune disease.

The compound is selected from a group consisting of compounds that bindcomponents of the immune complex and either prevent its formation orprevent the autoantigen from activating a Toll-like receptor (TLR). Suchcompounds include Toll-like receptor decoys, compounds that inhibit theactivity of MyD88, compounds that inhibit production of immune complexcomponents (e.g., antisense nucleotides), dominant-negative TLR, aToll-like receptor antagonist, and compounds that inhibit signalingpathways activated by the interaction or binding of the immune complexor autoantigen to the TLR. Preferably, the compound binds and/orinhibits function of Toll-like receptors TLR2, TLR3, and TLR9 orfunctional domains thereof. More preferably, the TLR is TLR3 or TLR9 ora functional fragment thereof.

Compounds that bind components of the immune complex and prevent itsformation or prevent binding of the complex to the Toll-like receptorand compounds that inhibit MyD88 signaling include polyclonal andmonoclonal antibodies, dominant negative proteins that can blockwildtype TLR activity or block components of the TLR-mediated signalingcascade, inhibitory oligodeoxynucleotides (ODN), such as S-ODN 2088(Lenart, et al., Antisense Nucleic Acid Drug Dev. 4, 247-256 (2001)),antisense nucleotides including RNA and modified nucleotides, or otherpathway specific kinase inhibitors. The compound is a compound otherthan chloroquine.

In one embodiment the invention provides methods for screening compoundsor agents that inhibit immune complex formation or binding to theToll-like receptor and/or inhibit B cell/dendritic cell activation. Themethods comprise contacting immune complex components with a test agentand measuring B cell or dendritic cell activation and/or proliferationand/or binding of the complex to the Toll-like receptor.

In an other embodiment, a method of diagnosing an ICAD is provided. Themethod comprises taking a biological sample comprising IgG of anindividual suspected of having ICAD, incubating the biological sampletogether with RF+ B cells or dendritic cells, and measuring theactivation of the RF+ B cells or dendritic cells, wherein a change inactivity in the RF+ B cell or dendritic cell cultures exposed to thebiological sample from the individual suspected of having ICAD relativeto the RF+ B cells or dendritic cells exposed to a biological samplecomprising IgG from a control individual is indicative of ICAD.Preferably, the change is an increase in activity. Activation of the Bcells can be measured, for example by measuring proliferation orupregulation of co-stimulatory molecules such as CD80 and CD86 as wellas upregulation of MHC class II molecules. Activation of dendritic cellscan be assessed by measuring the expression of co-stimulatory molecules,production of cytokines, e.g., TNF-α, or changes in the dendritic cellphenotype.

In yet another embodiment, the invention provides an in vivo modelsystem for evaluating a compound or an agent for its efficacy intreating ICAD. The model system comprises administering a test agent toan ICAD model animal including mouse and rat models, and measuring Bcell or dendritic cell activation and/or proliferation and/or binding ofthe complex to the Toll-like receptor in such animal, wherein decreasedactivation is indicative of an agent which is capable of treating ICAD.Alternatively, one can use cell lines expressing Toll-like receptors andscreen for compounds that modulate, preferably inhibit or block, suchreceptors.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with the description, serve to explain and illustrate theobjects, advantages, and principles of the invention. In the drawings,

FIG. 1 shows that anti-nucleosome antibody (PL2-3) stimulation of AM14RF+ B cells is DNase sensitive. The IgG2a anti-nucleosome antibody bindsto chromatin released from the B cells in culture to form an immunecomplex which is recognized by the B cell via it's IgG2a-specificantigen receptor. AM14 RF+ spleen cells were pre-incubated with variousconcentrations of DNase I (0, 5, 15, or 50 μg/ml) for 15 minutes priorto the addition of goat anti-mouse IgM F(ab′)₂, PL2-3 (IgG2a^(j)anti-nucleosome mAb), or LPS. Results are expressed as the percentage ofthe maximum response to each ligand in the absence of DNase.

FIGS. 2A-2B show that anti-TNP/TNP-BSA IC fail to efficiently stimulateAM14 RF+ B cell proliferation. The anti-TNP mAbs Hy1.2 (IgG2a^(a)) andC4010 (IgG2a^(b)) were mixed with varying concentrations of TNP-BSA [50μg/ml (circle), 12.5 μg/ml (square), 3.1 μg/ml (up-triangle)] to form ICwith antibody/protein ratios of 1:1, 4:1, and 16:1 respectively. In FIG.2A, IC formation was confirmed by a shift in Clq binding activityrelative to uncomplexed antibodies (down-triangle). In FIG. 2B theability of the anti-TNP/TNP-BSA IC described above to stimulate RF+ Bcell proliferation was compared to the stimulation induced by theanti-nucleosome mAb PR1-3, uncomplexed Hy1.2, C4010, or 50 μg/mlTNP-BSA.

FIG. 3 shows that autoantibody/autoantigen-IC stimulation of RF+ B cellsis not complement receptor dependent. Spleen cells from each of two WTcontrol mice (gray), RF+CR+/+ mice (white), and RF+CR−/− mice (hatched)were stimulated with goat anti-mouse IgM F(ab′)₂, CpG S-ODN 1826, theanti-nucleosome mAbs PR1-3 (IgG2a^(j)), PL2-3 (IgG2a^(j)), PL2-8(IgG2b), or 3% serum from representative autoimmune mice (lpr/gld andgld).

FIGS. 4A-4C show that autoAb/autoAg-IC stimulation of RF+ B cells isMyD88-dependent. In FIG. 4A, T-depleted spleen cells from wildtype (WT)and RF+ MyD88−/− mice were stained with B220 and an idiotype specificantibody, 4G7. In FIG. 4B, MyD88 WT or knock-out mice were identified byPCR. In FIG. 4C spleen cells from each of two WT (gray), RF+ MyD88+/+(white), RF+ MyD88−/− (hatched) mice were stimulated with anti-IgMF(ab′)₂, CpG S-ODN 1826, LPS, anti-nucleosome mAbs PR1-3, PL2-3, PL2-8,or 3% lpr/gld serum. Total cpm for the anti-IgM stimulated cultures were83,414/39,049 (WT); 93,126/61,315 (RF+ MyD88+/+); and 39,826/57,484 (RF+MyD88−/−). Results are expressed as the percentage of the anti-IgMresponse and are representative of four separate experiments.

FIGS. 5A-5C demonstrate that autoAb/autoAg-IC stimulation of RF+ B cellscan be blocked by inhibitors of the TLR9 signaling pathway. In FIG. 5A,RF+ B cells were preincubated for 15 minutes with 1 or 2 μg/mlchloroquine (a), preincubated for 2 hrs with concanamycin B (b), orpreincubated for 30 minutes with inhibitory CpG S-ODN 2088 (c), prior tothe addition of the stimulatory ligands anti-IgM F(ab′)₂, 5-50 μg/mlPL2-3, 0.3-2.0 μg/ml CpG S-ODN 1826, LPS, lipopeptide, or porin B. InFIG. 5B, RF+ B cells were preincubated for 30 min with 12 μg/ml CpGS-ODN 2088 prior to the addition of stimulatory ligands. Results areexpressed as a percentage of the anti-IgM response in the absence ofinhibitor. The data in (FIG. 5A) and (FIG. 5C) is representative of 2-4experiments, while (FIG. 5B) is the mean of two experiments.

FIGS. 6A-B show the results of an experiment where MyD88-deficient(MyD88−/−) Fas-sufficient mice were bred with MyD88-sufficient(MyD88+/+) Fas-deficient (lprlpr) autoimmune mice to generate MyD88−/−lpr/lpr mice, and appropriate control groups. Anti-nuclear-antibodies(ANA) in the sera of age-matched MyD88+/+ lprlpr (FIG. 6A) and MyD88−/−lpr/lpr (FIG. 6B) littermates (12-13 weeks of age) were detected byindirect immunofluorescence using HEp-2 cells as substrate as previouslydescribed (Rifkin et al., Journal of Immunology 161: 5164-5170, 1998).Serum from a diseased autoimmune MRL-lpr/lpr mouse served as thepositive control and serum from a non-autoimmune BALB/c mouse as thenegative control. This in vivo experiment demonstrates that, in astandard and widely used lupus mouse strain, autoantibody productionrequires signaling through MyD88. This strongly suggests that TLRactivation is required for the development of lupus in this model.

FIGS. 7A-7B show that B cells can be activated through TLR9 and thatthis activation can be blocked by TLR9 inhibition with ODN 2088. B cellswere purified from the spleen of MRL+/+ mice and either pre-incubated,or not pre-incubated, for 30 minutes with varying doses of ODN 2088, aninhibitory oligodeoxynucleotide that specifically blocks signalingthrough TLR9. Anti-IgM F(ab′)₂ (Anti-IgM), that activates through the Bcell receptor (7A), or the stimulatory ODN 1826, that specificallyactivates through TLR9 (7B), was then added to the cultures. After 20-24hours, ³[H] thymidine was added to the cultures and, after an additional14-18 hours, proliferation was determined by measuring thymidineincorporation using an LKB Wallac 1212 Rackbeta counter. Onlystimulation through TLR9 is blocked by ODN 2088 demonstrating thespecificity of the inhibitory effect: stimulation induced by B cellreceptor cross-linking is not inhibited.

FIGS. 8A-8C show that dendritic cells can be specifically activatedthrough TLR9 and this activation can be blocked by TLR9 inhibition withODN 2088.

FIG. 9 shows the inhibitory effects of ODN 2088 on chromatin-containingimmune complex mediated B cell proliferation are long-lasting. B cellswere purified from the spleen of MRL+/+ mice and either pre-incubated,or not pre-incubated, for 16-20 hours with 12 μg/ml ODN 2088. After thispre-incubation, the cells were washed to remove ODN 2088 from thecultures and then the B cells were cultured with either medium alone orwith fresh ODN 2088 at 12 μg/ml. Thirty minutes later the anti-chromatinmonoclonal antibody PL2-3 (50 μg/ml) or LPS (10 μg/ml) were added to thecultures. After 20-24 hours, ³[H] thymidine was added to the culturesand, after an additional 14-18 hours, proliferation was determined bymeasuring thymidine incorporation using an LKB Wallac 1212 Rackbetacounter.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for treatingand/or preventing immune complex associated diseases (ICAD), in asubject having an ICAD or at risk for the ICAD.

For the purposes of this invention the term “immune complex associateddiseases” or “ICAD” refers to diseases including, but not limited tosystemic lupus erythematosus (SLE) and related connective tissuediseases, rheumatoid arthritis, hepatitis-C and hepatitis B relatedimmune complex disease (e.g., cryoglobulinemia), Behcets disease,autoimmune glomerulonephritides, and vasculopathy associated with thepresence of LDL/anti-LDL immune complexes.

We have discovered that immune complexes (IC) containing an autoantigen,such as chromatin, but not IC containing a foreign antigen, are able toactivate autoreactive B cells and that this activation is dependent onthe ability of the autoantigen-containing IC to sequentially engage boththe B cell receptor and a second receptor, a Toll-like receptor. Thisfinding establishes a novel link between the innate and adaptive immunesystems and consequently a general mechanism whereby autoreactive Bcells specific for protein/nucleic acid autoantigens are activated.

The Toll-like receptors (TLRs) are a family of membrane bound receptorproteins that are critically involved in innate immune responses andrecognize pathogen associated molecular patterns or determinants thatappear unique to microorganisms and are involved in activating immunecells against the source of these microbial particles.

The Toll-like family of receptors have leucine-rich repeats in theirextracellular domains and the Toll/IL-1 receptor homology domain intheir cytoplasmic domains. The Toll family is remarkably conserved inevolution. The first member discovered was the product of the toll genewhich is part of the signaling pathway responsible for the specificationof dorsal-ventral polarity in the early development of the fruit flyDrosophila melanogaster.

Currently ten mammalian homologues have been identified, called TLR1through TLR10. Both the IL-1 receptor and the TLRs share similardownstream effects, such as the activation of immune response genes, andall these receptors work through signaling cascades that include theadaptor protein MyD88 (Mussio et al., Science 278:1612; Wesche et al.,Immunity 7:837) which has been previously described as a myeloiddifferentiation protein (Lord et al., Oncogene 5:1095). In general, thedifferent TLRs are thought to be activated by different types ofmicrobial particles (Hemmi et al., Nature 408:740-745 (2000); Underhillet al., Nature 402:39-43 (1999); Aliprantis et al., EMBO J.,19:3325-3336 (2000)). However, there is also accumulating evidence thatin addition to microbial particles, mammalian TLRs can also recognizecertain self (mammalian) antigens, in particular cytoplasmic componentsthat are released from cells as a result of cell death (Akira et al.,Nat. Immunol., 2: 675-680 (2000).

The term “Toll-like receptor” is herein meant to include an intactToll-like receptor, for example a receptor that has been described inthe Online Mendelian Inheritance in Man under access numbers 601194TOLL-LIKE RECEPTOR 1, TLR1; 603028 TOLL-LIKE RECEPTOR 2, TLR2; 603029TOLL-LIKE RECEPTOR 3, TLR3; 603030 TOLL-LIKE RECEPTOR 4, TLR4; 603031TOLL-LIKE RECEPTOR 5, TLR5; 605403 TOLL-LIKE RECEPTOR 6, TLR6; 300365TOLL-LIKE RECEPTOR 7, TLR7; 300366 TOLL-LIKE RECEPTOR 8, TLR8; 605474TOLL-LIKE RECEPTOR 9, TLR9; and 606270 TOLL-LIKE RECEPTOR 10, TLR10 or afunctional fragment thereof such as, for example, a soluble form of theToll-like receptor, i.e. where the membrane binding domain has beendeleted or altered, in some embodiments the cytoplasmic domain is alsonot present, or a MyD88 binding or interacting fragment of the Toll-likereceptor or a homolog of the Toll-like receptor capable of binding to orinteracting with MyD88. More preferably the TLR is TLR9.

Our experiments to this point have identified the importance of theinteraction between TLR9 and the chromatin component of thechromatin-containing immune complexes. However, in SLE and relateddiseases immune complexes commonly form with autoantibodies and distinctRNA/protein antigenic complexes including the Sm antigen and smallnuclear ribonucleoproteins (snRNP) (Tan, E. Adv. Immunol. 44, 93-151(1989)). In addition, in immune complex associated diseases such ashepatitis C, pathogenic immune complexes form between antibodies and theRNA containing viruses. TLR3 has been identified as a specific receptorfor double-stranded RNA (Alexopoulou, Nature 413, 732-738 (2001). Thus,it is reasonable to anticipate that TLR3 engagement will be important inthe activation process elicited by these RNA-containing immunecomplexes.

Preferably the TLR is TLR3 or TLR 9, or a functional fragment thereof ora fragment that is homologous to TLR 3 or TLR 9 and capable of bindingor interacting with MyD88.

The Toll-like receptor useful according to the present invention canalso be a fusion receptor wherein the Toll-like receptor is fused withanother protein such as a Myc-tag. The preferred Toll-like receptorsinclude Toll-like receptor-2 or Toll/Interleukin-1 receptor-like 4(Chaudhary, et al., Blood 91: 4020-4027, 1998), Toll-like receptor-3(Rock, et al., Proc. Nat. Acad. Sci. 95: 588-593, 1998), and Toll-likereceptor-9 (Chuang and Ulevitch, Europ. Cytokine Netw. 11: 372-378,2000; Du, et al., Europ. Cytokine Netw. 11: 362-371, 2000) or functionalfragments thereof or homologs that have a similar function, such as forexample, binding MyD88.

As used herein, the terms “treatment” or “treating” include: (1)preventing such disease from occurring in a subject who may bepredisposed to these diseases but who has not yet been diagnosed ashaving them; (2) inhibiting these diseases, i.e., arresting theirdevelopment; or (3) ameliorating or relieving the symptoms of thesediseases, i.e., causing regression of the disease states.

The compounds preferably inhibit activation of B cells (BC) or dendriticcells (DC) by at least about 50% in an in vitro or in vivo assaysdiscussed below. More preferably the compounds inhibit autoantigenicactivation of BCs or DCs via the Toll-like receptor by 75%, mostpreferably 95%. Additional compounds are identified and tested in thescreening assays discussed in more detail below.

The compound useful according to the present invention is selected froma group consisting of compounds that bind components of the immunecomplex or Toll-like receptors. These compounds either prevent formationof the immune complex; prevent the autoantigen or the autoantigenicfragment of the immune complex from activating a Toll-like receptor(TLR) or prevent the downstream molecular signaling, such as thatmediated through MyD88, from the Toll-like receptor. Such compoundsinclude Toll-like receptor decoys, compounds that inhibit the activityof MyD88, compounds that inhibit production of immune complex components(e.g., antisense nucleotides)), dominant-negative TLR, a Toll-likereceptor antagonist or blocker (such as ODN 2088 or antibodies againstthe TLRs), and compounds that inhibit signaling pathways activated bythe interaction or binding of the autoantigenic fragment of the immunecomplex to the TLR. Preferably, the compound binds and/or inhibitsfunction of Toll-like receptors TLR2, TLR3, and TLR9 or functionaldomains thereof. More preferably, the TLR is TLR3 or TLR9 or afunctional fragment thereof.

The invention further provides efficient screening methods to identifypharmacological agents or lead compounds for agents which inhibitbinding of the immune complex (or the autoantigenic component thereof)to the TLR, preferably TLR2, TLR3, TLR9, or any combination thereof,most preferably TLR3 or TLR9. The methods are amenable to automated,cost-effective high throughput drug screening and have immediateapplication in a broad range of pharmaceutical drug developmentprograms.

Generally, these screening methods involve assaying for compounds thatmodulate, for example, the autoantigen interaction with a TLR,preferably TLR2, TLR3, TLR9, or any combination thereof, most preferablyTLR3 or TLR 9. Still, more preferably the compound modulates interactionwith TLR 9. A wide variety of assays for binding agents are providedincluding labeled in vitro protein-protein binding assays, immunoassays,cell based assays, etc.

In vitro binding assays employ a mixture of components including a TLRpolypeptide, preferably TLR2, TLR3, TLR9, or any combination thereof,most preferably TLR3 or TLR9, still more preferably TLR9 which may bepart of a fusion product with another peptide or polypeptide, e.g. a tagfor detection or anchoring, etc. The assay mixtures may also comprise anatural intracellular TLR binding target, such as MyD88. While nativefull-length binding targets may be used, it is frequently preferred touse portions (e.g. peptides) thereof so long as the portion providesbinding affinity and avidity to the subject MyD88 polypeptideconveniently measurable in the assay. The assay mixture also comprises acandidate pharmacological agent. Candidate agents encompass numerouschemical classes, though typically they are organic compounds;preferably small organic compounds and are obtained from a wide varietyof sources including libraries of synthetic or natural compounds. Avariety of other reagents may also be included in the mixture. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, protease inhibitors, nuclease inhibitors, antimicrobialagents, etc. may be used.

The resultant mixture is incubated under conditions whereby, but for thepresence of the candidate pharmacological agent, the TLR polypeptidespecifically binds the cellular binding target, portion or analog with areference binding affinity. The mixture components can be added in anyorder that provides for the requisite bindings and incubations may beperformed at any temperature that facilitates optimal binding.Incubation periods are likewise selected for optimal binding but alsominimized to facilitate rapid, high-throughput screening.

After incubation, the agent-biased binding between the TLR polypeptideand one or more binding targets is detected by any convenient way. Adifference in the binding affinity of the TLR polypeptide to the targetin the absence of the agent as compared with the binding affinity in thepresence of the agent indicates that the agent modulates the binding ofthe TLR polypeptide to the TLR binding target. Analogously, in thecell-based assay also described below, a difference in TLR-dependenttranscriptional activation in the presence and absence of an agentindicates the agent modulates TLR function, for example, binding toMyD88. A difference, as used herein, is statistically significant andpreferably represents at least a 50%, more preferably at least a 75%,still more preferably at least a 90% difference.

The preferred assay to test a pharmaceutical agent for potency ofinterrupting the activation of B cells or dendritic cells comprises RF+B cells or dendritic cells which are incubated with a test agent and aTLR stimulatory agent, such as PR1-3, PL2-3, or a CpG S-ODN 1826 (see,e.g. Leadbetter et al., Nature, 416:603-607, 2002) in a suitable cellculture medium. Incubation with the test agent can be performed prior toor simultaneously with the addition of the stimulatory agent.

The activation of the B cells can be observed using a number oftechniques well known to one skilled in the art. For example, the ³[H]thymidine incorporation assay can be performed to measure proliferationof B cells. Alternatively, activation of B cells can be analyzed usingfluorescence activated cell sorting (FACS) to measure cell surfaceexpression of, for example, CD80, CD86 or MHC class II molecules.Activation of dendritic cells can be observed by measuring production ofcytokines, such as TNF-α, interferon-α, IL-12 and BAFF, orco-stimulatory molecules such as CD80, CD86 or MHC class II molecules(Banchereau, et al., Annu. Rev. Immunol. 18: 767-811, 2000; Mackay andBrowning, Nat. Rev. Immunol. 2: 465-475).

Alternatively, dendritic cell activation can be observed by measuringmorphologic changes in the DC (Banchereau, et al., Annu. Rev. Immunol.18: 767-811, 2000).

If the stimulation of the expression of, for example a co-stimulatorymolecule is decreased as a result of adding the test agent, the testagent is considered to be a potential agent to treat ICAD. Theexpression is considered decreased if it is at least about 50%, morepreferably at least about 60-75%, most preferably at least about 90%decreased as compared to a cell sample where no test agent has beenadded. More detailed examples are given in the Examples in the end ofthis section.

A preferred assay mixture of the invention is set forth in the Examples.An assay mixture of the invention also comprises a candidatepharmacological agent. Generally, a plurality of assay mixtures are runin parallel with different candidate agent concentrations to obtain adifferential response to the various concentrations. Typically, one ofthese assay mixtures serves as a negative control, i.e. at zeroconcentration or below the limits of assay detection. Candidate agentsencompass numerous chemical classes, though typically they are organiccompounds and preferably small organic compounds. Small organiccompounds suitably may have e.g. a molecular weight of more than about50 yet less than about 2,500. Candidate agents may comprise functionalchemical groups that interact with proteins and/or DNA.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and peptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Libraries can be composed of smallpolypeptides (see, for instance, Lam et al., Nature, 354: 82, 1991 andWO 92/00091; Geysen et al., J Immunol Meth, 102: 259, 1987: Houghten etal., Nature, 354: 84, 1991 and WO 92/09300 and Lebl et al., Int J PeptProt Res, 41, 201, 1993). Alternatively, libraries of small non-peptidemolecules can be based upon a common template or core structure (see,for instance, Ellman and Bunin, J Amer Chem Soc, 114:10997, 1992 forbenzodiazepine template; WO 95/32184 for oxazolone and aminidinetemplate; WO 95/30642 for dihydrobenzopyran template and WO 95/35278 forpyrrolidine template.

Additionally, natural and synthetically produced libraries and compoundsare readily modified through conventional chemical, physical, andbiochemical means. In addition, known pharmacological agents may besubject to directed or random chemical modifications, such as acylation,alkylation, esterification, amidification, etc.

Antibodies and binding fragments thereof or aptamers (available from,for example SomaLogic Inc., Boulder, Colo.) that bind immune complexcomponents and prevent formation or TLR binding are also useful.Examples of antibodies useful according to the invention includemonoclonal antibodies against TLRs such as anti-TLR3 monoclonal antibody(Matsumoto et al., Biochem. Biophys. Res. Commun., 293:1364-1369, 2002)anti-TLR-9 (Toll-like receptor 9, Imgenex, San Diego, Calif.) orhumanized versions thereof.

The term dominant negative Toll-like receptor as used herein refers toToll-like receptors, that have been engineered to have a defect, such asdeletion of the domain required for downstream signaling but which canbind the antigenic portion of the immune complex.

The antibodies and binding fragments thereof can be either polyclonal ormonoclonal, but preferably are monoclonal. If polyclonal, the antibodiescan be in the form of antiserum or monospecific antibodies, such aspurified antiserum which has been produced by immunizing animals withpurified protein. Preferably, however, the antibodies are monoclonalantibodies so as to minimize the administration of extraneous proteinsto an individual. Monoclonal antibodies can be prepared according towell known protocols. See. e.g., Skare et al., J. Biol. Chem. 268:16302-16308 (1993); and U.S. Pat. Nos. 4,918,163 and 5,057,598, whichare incorporated herein by reference. The antibodies can be whole,Fab's, single chain, single domain heavy chain, etc. Single chainantibodies are preferable. Methods for the production of single chainbinding polypeptides are described in detail in, e.g., U.S. Pat. No.4,946,778, which is incorporated herein by reference.

When an antibody or other protein or peptide is used the peptide ispreferably conjugated to a carrier such as biotin or a poly(alkalineoxide), for example polyethylene glycol (PEG). Polymeric substances suchas dextran, polyvinyl pyrrolidones, polysaccharides, starches, polyvinylalcohols, polyacryl amides or other similar polymers can be used. Thepoly(alkaline oxide) can include monomethoxy polyethylene glycol,polypropylene glycol, block copolymers of polyethylene glycol and thelike. Polyethylene glycol as poly(alkylene oxide) is preferred. Thepolymers can also be distally capped with C₁₋₄ alkyls instead ofmonomethoxy groups.

For administration to humans, e.g., as a component of a composition forin vivo treatment, the monoclonal antibodies are preferablysubstantially human or humanized to minimize immunogenicity, and are insubstantially pure form. By “substantially human” is meant that theimmunoglobulin portion of the composition generally contains at leastabout 70% human antibody sequence, preferably at least about 80% human,and most preferably at least about 90-95% or more of a human antibodysequence.

For therapeutic applications, the compounds may be suitably administeredto a subject such as a mammal, particularly a human, alone or as part ofa pharmaceutical composition, comprising the compounds together with oneor more acceptable carriers thereof and optionally other therapeuticingredients. The carrier(s) must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

The pharmaceutical compositions of the invention include those suitablefor oral, rectal, nasal, topical (including buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenousand intradermal), ocular using eye drops, transpulmonary usingaerosolubilized or nebulized drug administration. The formulations mayconveniently be presented in unit dosage form, e.g., tablets andsustained release capsules, and in liposomes, and may be prepared by anymethods well know in the art of pharmacy. (See, for example, Remington:The Science and Practice of Pharmacy by Alfonso R. Gennaro (Ed.) 20thedition, Dec. 15, 2000, Lippincott, Williams & Wilkins; ISBN:0683306472.)

Such preparative methods include the step of bringing into associationwith the molecule to be administered ingredients such as the carrierwhich constitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing intoassociation the active ingredients with liquid carriers, liposomes orfinely divided solid carriers or both, and then if necessary shaping theproduct.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion, or packed in liposomes and as a bolus,etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets optionally may be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Compositions suitable for topical administration include lozengescomprising the ingredients in a flavored basis, usually sucrose andacacia or tragacanth; and pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia.

Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.

It will be appreciated that actual preferred amounts of a given compoundused in a given therapy will vary according to the particular compoundbeing utilized, the particular compositions formulated, the mode ofapplication, the particular site of administration, the patient'sweight, general health, sex, etc., the particular indication beingtreated, etc. and other such factors that are recognized by thoseskilled in the art including the attendant physician or veterinarian.Optimal administration rates for a given protocol of administration canbe readily determined by those skilled in the art using conventionaldosage determination tests.

The present invention also provides methods for diagnosing ICAD in anindividual. The method comprises of measuring the Toll-like receptormediated activation of a B cell or dendritic cell induced by abiological sample containing an immune complex(es) consisting ofautoantigen(s) and autoantibody(ies). Activation of a Toll-like receptorin B cells can be measured indirectly, for example by measuringproliferation of the B cells, expression of various co-stimulatorymolecules such as CD80, CD86 or upregulation of MHC class II molecules.Activation of Toll-like receptor in dendritic cells can be measured byobserving morphologic changes of the dendritic cells either with orwithout specific staining, or by measuring expression of co-stimulatorymolecules such as CD80 and CD86, or by measuring upregulation ofactivation markers such as CD69, or by measuring upregulation ofchemokine receptors such as CCR7, or by measuring the production ofcytokines such as TNF-α using a number of different techniques includingassaying mRNA or protein levels.

For example, change in the expression of TNF-α can be measured using RNAisolated from dendritic cells. RNA quantitation methods includepolymerase chain reaction (PCR) based methods such as, for example, aTaqMan® system (Applied Biosystems), Brilliant Quantitative PCR(Stratagene), Platinum® quantitative PCR (Resgen, Inc.). RNA is isolatedfrom a biological sample, for example blood sample, which is taken froman individual suspected of having ICAD. The amount of Toll-like receptormRNA is quantified using, for example, techniques listed above andcompared to a sample from a control individual.

Preferably, assays such as FACS analysis are used to measure proteinexpression of the co-stimulatory molecules. If the expression of theseco-stimulatory molecules is increased, it indicates that the individualis affected with an ICAD. The activity is considered increased if theassay shows at least about 5% increase in the amount of theco-stimulatory molecule compared to the control sample.

Alternatively, serum from a test individual suspected of having ICAD canbe injected to a cell culture expressing a functional Toll-like receptorpathway. The activity of the Toll-like receptor is consequently measuredfrom the cell culture treated, in parallel, with control and testsubject serum. If the activity of the Toll-like receptor is increased inthe cells treated with the test individual's serum compared to the cellstreated with the control serum, it indicates that the individual isaffected with an ICAD. The expression is considered increased if it isat least about 5% higher than in the control sample.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modification within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

EXAMPLE 1

Autoreactive B cells are present in the lymphoid tissues of healthyindividuals, but typically remain quiescent. When this homeostasis isperturbed, the formation of self-reactive antibodies can have seriouspathological consequences. B cells expressing an antigen-receptorspecific for self-IgG make a class of autoantibodies known as rheumatoidfactor (RF). Here we show that effective activation of RF+ B cells ismediated by IgG2a/chromatin immune complexes and requires the sequentialengagement of the antigen-receptor and a MyD88-dependent Toll-likereceptor family member. These data establish a critical link between theinnate and adaptive immune systems in the development of systemicautoimmune disease and explain the preponderance of autoantibodiesreactive with nucleic acid/protein particles. The unique features ofthis dual-engagement pathway should facilitate the development oftherapies that specifically target autoreactive B cells.

We have now developed a model in vitro system for analyzing the factorsinvolved in the activation of autoreactive B cells and dendritic cellsin autoimmune disease. One of the prevalent autoantibody specificitiesin lupus-prone lpr mice is rheumatoid factor (RF). RFs are antibodiesthat are reactive with self IgG. RF B cells become activated, expand innumber, and differentiate into antibody secreting cells in autoimmunestrains of mice, but not in mice of a non-autoimmune background. Inorder to investigate the mechanisms involved in this pathologicactivation, we studied primary RF+ B cells from the spleens of micegenetically engineered to express a pair of transgenes which wouldconfer a particular RF specific to most B cells. These B cells areprototypes of the autoreactive B cells found in SLE patients.

Initially, we demonstrated that these RF+ B cells were activated byserum samples from autoimmune mice, but not by serum form non-autoimmunemice (Rifkin et al, 2000). We further proved that the stimulatory factorin the serum was, in fact, IgG2a. However, comparable amounts of IgG2aisolated from non-autoimmune sera were not stimulatory, indicating thatnot all IgG2a was sufficient to stimulate RF+ B cells. Therefore weconcluded that IgG2a in the autoimmune sera had unique properties. Tofurther identify the relevant IgG2a component, we used IgG2a monoclonalantibodies (mAbs) and showed that mAbs specific for self-antigens, suchas DNA-histone complexes, were stimulatory while antibodies specific forforeign or non-self antigens were not stimulatory.

We next considered the possibility that autoantibody/autoantigen immunecomplexes were the relevant component. When the assays with sera andmAbs were run in the presence of DNAse, stimulation of the RF+ B cellswas dramatically reduced (FIG. 1). These data suggested that cleavage ofthe DNA backbone results in the dissociation of theautoantibody/autoantigen (DNA) immune complex. This strongly indicatesthat immune complexes of autoantigens/autoantibodies have uniqueproperties which preferentially activate autoreactive B cells. Theinability of immune complexes containing non-self antigen to activateautoreactive B cells is shown in FIG. 2.

We investigated the possibility that autoantibody/autoantigen immunecomplexes are capable of engaging a second receptor, which thenfacilitates the activation of the RF B cells. Since recent studies haveshown that a family of proteins known as Toll-like receptors (TLR) canbind to bacterial DNA and lead to macrophage, dendritic cell and B cellactivation, we considered the possibility that TLR could play a role inthe activation of these autoreactive RF B cells. TLR are currently avery popular area of study, and have been shown to be responsible tobinding and recognition of many pathogen-associated molecular patterns(PAMPS), so this link to innate immune responses would be a very uniqueconnection for autoimmunity. All known mammalian TLR signal through theadaptor protein, MyD88. TLR-mediated activation in mice in which theMyD88 gene has been eliminated by genetic engineering is severelycompromised and signals mediated through TLR9 are completely abolished.We bred our transgenic RF+ mice onto a MyD88−/− background. Theresulting mice were positive for the RF transgenes (FIG. 4A) and lackedMyD88 signaling molecules (FIG. 4B).

We found that RF MyD88−/− mice fail to respond to any of theautoantibodies which otherwise stimulate proliferation of RF MyD88+/+ Bcells (see FIG. 4C). We tested multiple forms of autoantibodies: serum,antinucleosome antibodies and anti-Sm antibodies. This is consistentwith the hypothesis that autoantibody/autoantigen immune complexescontain repeating pattern components such as DNA or RNA which engageToll receptors either inside the B cell (DNA/TLR9) or on the surface ofthe B cell (RNA/TLR3). In the case of autoreactive RF+ B cells, theautoantigen/autoantibody immune complexes likely sequentially engage theBCR and the TLR, leading to increased activation and proliferation. Thisis the first demonstration that co-engagement of the BCR and TLR canresult in the activation of autoreactive B cells. This connectionprovides a very unique link between autoimmunity and the body's innateimmune response and may provide an initiating mechanism for many of thepathogenic responses in autoimmune diseases such as SLE and rheumatoidarthritis.

Subsequent studies demonstrated that drugs known to block B cellactivation by stimulatory CpG ODN via TLR9, such as chloroquine andconcanamycin A, completely blocked the ability of the chromatincontaining IC to stimulate RF+ B cells. The response could also beblocked by inhibitory CpG ODN which specifically inhibits activationthrough TLR9. These data demonstrate that TLR9 is a key receptor in theactivation of autoreactive B cells. The data demonstrate that the IgGcomponent of the immune complex binds to the BCR leading to theinternalization of the immune complex and delivery to an internalvesicular compartment. The subsequent engagement of TLR9 by thechromatin component of the IC within this internal compartment leads toB cell activation. Factors that contribute to the enhanced availabilityand thus uptake of chromatin per se (susceptibility factors for SLE)could also lead to B cell activation through a similar mechanism. It isof note that anti-chromatin antibodies are the first detectableautoantibody present in the sera of SLE patients and animals models ofspontaneous SLE.

EXAMPLE 2

Patients afflicted with SLE and other systemic autoimmune diseasesproduce a wide range of autoantibody specificities. Very frequentlythese autoantibodies bind to chromatin or other subcellular nucleicacid/protein particles (Tan, E. Adv. Immunol. 44, 93-151 (1989)).MRL/lpr mice, a well-studied murine model of systemic lupuserythematosus and rheumatoid arthritis, also produce exceedingly hightiters of IgG anti-IgG rheumatoid factor (RF) (Theofilopoulos, et al.,J. Exp. Med. 162, 1-18 (1985); Wolfowicz, et al., Clin. Immunol.Immunopath. 46, 382-395 (1988)). This RF response has provided a highlyrelevant transgenic model for the study of autoantibody regulation. Bcells from the AM14 transgenic mouse line express an antigen receptorspecific for IgG2a^(a/j), originally captured as a hybridoma productfrom the spleen of a diseased MRL/lpr mouse (Shlomchik, et al., Int.Immunol. 5, 1329-1341 (1993)). The AM14 allotype specificity andrelatively low affinity for monomeric IgG2a are typical of the diseaseassociated RF repertoire (Jacobson, et al., J. Immunol. 152, 4489-99(1994)). In wildtype mice, AM14 RF+ B cells develop normally and remainfunctionally naïve (Hannum, et al., J. Exp. Med. 184, 1269-1278 (1996)).However, on an autoimmune-prone lpr background of the cognate allotype,they become activated, proliferate and secrete autoantibody (Wang, andShlomchik, J. Exp. Med. 190, 639-649 (1999)). A number of factors arelikely to contribute to the activation of AM14 B cells in lpr mice, notthe least of which is the status of the autoantigen, IgG2a.

RF+ B Cells are Activated by Autoantibody/Autoantigen Immune Complexes

We have previously shown that AM14 RF+ B cells can be activated in vitroby IgG2a^(a/j) isolated from the sera of autoimmune mice, but not bycomparable levels of IgG2a present in sera obtained from wildtype mice(Rifkin, et al., J. Immunol. 165, 1626-1633 (2000)). RF+ B cells werealso found to proliferate strongly in response to affinity purifiedIgG2a^(j) mAbs specific for nucleosomes, a self-antigen, whereasIgG2a^(a/j) mAbs specific for haptens or other foreign antigens producedlittle, if any, response (Rifkin, et al., J. Immunol. 165, 1626-1633(2000)). These studies suggested that immune complexes (IC) formedbetween IgG2a nucleosome-specific mAbs and chromatin fragments releasedfrom co-cultured cells (Emlen, et al., J. Immunol. 148, 3042-3048(1992)) could effectively activate RF+ B cells, while monomericanti-hapten IgG2a antibodies could not. To further test this premise,DNase was added to the assay medium as a means of disrupting theputative IC. The vigorous proliferative response normally elicited bythe purified anti-nucleosome mAb PL2-3 (Monestier and Novick, Mol.Immunol. 33, 89-99 (1996)) or by autoimmune serum was dramaticallyreduced (FIG. 1). Nonspecific toxicity of the DNase was not a factor asthe responses to F(ab′)₂ anti-IgM and LPS were unaffected (FIG. 1).These data confirm that RF+ B cells respond to IgG2a IC that containDNA, not to monomeric IgG2a antibodies alone.

If RF+ B cell activation by IgG2a IC simply resulted from more effectivecrosslinking of the antigen receptor than would be possible withmonomeric IgG2a, then conventional anti-hapten/hapten-protein complexesshould also strongly stimulate RF+ B cells. To address this issue, ICswere prepared by pre-incubating IgG2a monoclonal anti-TNP antibodieswith varying concentrations of TNP-BSA; complex formation was confirmedby demonstrating an increase in Clq binding activity (FIG. 2 a). Asexpected, IC of C4010, an IgG2a^(b) anti-TNP mAb, failed to stimulateRF+ B cells at all anti-TNP/TNP-BSA ratios. Surprisingly, IC of Hy1.2,an IgG2a^(a) anti-TNP mAb, only elicited a very modest proliferativeresponse relative to that elicited by anti-nucleosome mAb-containing IC.This discrepancy indicated that the extent of RF+ B cell proliferationelicited by an IC depends on both the antibody and the nature of the(auto)antigen.

Activation by IC is not Dependent on Complement Receptor Co-Engagement

A possible explanation for the dramatic difference in stimulatorycapacity of the anti-nucleosome and anti-TNP IC was that only the formercould synergistically engage both the B cell receptor (BCR) and a secondreceptor on the B cell surface. One potential candidate receptor was thecomplement receptor CD21, as complement components have been shown tobind autoantigen IC and could, in theory, serve to effectively co-engageCD19/CD21 and the BCR (Carter et al., J. Immunol. 141, 457-463 (1988)).However, when the AM14 heavy and light chain transgenics were bred ontomice deficient for CR1/2Cr2 (Ahearn, et al. Immunity 4, 251-262 (1996)),RF+ B cells from the CR1/2-deficient and CR1/2-sufficient littermatesresponded comparably to IgG2a anti-nucleosome mAbs and autoimmune sera(FIG. 3). B cells from control RF— littermates failed to respond to anyform of IgG2a, confirming the specific nature of the ligands used inthis study. A mitogenic hypomethylated CpG oligodeoxynucleotide wasincluded as a control stimulus. These data demonstrate that complementreceptors are not required for the RF+ B cell response toautoantibody/autoantigen IC.

Role of a MyD88-Dependent Receptor

Potential candidate receptors included members of the Toll-like receptor(TLR) family. These pattern recognition receptors were first describedin Drosophila where they were shown to trigger the release ofanti-fungal peptides (Lemaitre et al., Cell 86, 973-983 (1996)).Subsequently identified mammalian homologues were found to recognize aseries of conserved microbial products (i.e. LPS, microbiallipoproteins, and hypomethylated CpG DNA) referred to as pathogenassociated molecular patterns (PAMPS) (Medzhitov, et al., Nature 388,323-324 (1997); Akira, et al., Nat. Immunol. 2, 675-680 (2001)). Initialstudies focused on the effects of microbial products on cells of theinnate immune system where they were found to stimulate the release of awide range of inflammatory mediators (Akira, et al., Nat. Immunol. 2,675-680 (2001)). However, it was later shown that in addition toexogenous microbial ligands, TLRs could also recognize endogenousligands released from damaged or stressed mammalian cells (Li, et al.,J. Immunol. 166, 7128-7135 (2001)). All known mammalian TLRs signalingpathways use the adapter protein MyD88 (Akira, et al., Nat. Immunol. 2,675-680 (2001)), although in the case of TLR4, an additionalMyD88-independent pathway has been described (Horng, et al., Nat.Immunol. 2, 835-841 (2001)).

To investigate the potential role of TLRs in mediating RF+ B cellresponses to autoantibody/autoantigen IC, we crossed the AM14 transgenesonto a MyD88−/− background (Adachi, et al., Immunity 9, 143-150 (1998)).B cells from RF-MyD88+/+ (wildtype), RF+ MyD88+/+, and RF+ MyD88−/−offspring, identified by a combination of PCR and FACS analysis (FIGS.4A-4B), were assessed for their ability to respond to anti-IgM, CpG ODN,and LPS, as well as to a panel of anti-nucleosome mAbs and autoimmunesera. B cells from the MyD88-deficient mice responded normally toanti-IgM, but were unable to respond to stimulatory CpG DNA (Hacker, etal., J. Exp. Med. 192, 595-600 (2000)). Their response to LPS was muchlower than in wildtype mice, but still detectable, consistent withpartial activation through MyD88, and responded only weakly to LPS,which is known to stimulate partially through a MyD88-independentmechanism (Horng, et al., Nat. Immunol. 2, 835-841 (2001)) (FIG. 4C).Most dramatically, B cells from the RF+ MyD88-deficient mice werecompletely unresponsive to the anti-nucleosome mAbs PR1-3 or PL2-3 or toany of the autoimmune sera that effectively stimulated RF+ MyD88+/+ Bcells (FIG. 4C). These results clearly demonstrate thatautoantibody/autoantigen IC are particularly stimulatory for RF+ B cellsbecause they synergistically engage both the BCR and a MyD88-dependentreceptor.

Hypomethylated CpG motifs are a common feature of bacterial DNA, butthey are also present in mammalian DNA promoter elements (Singal andGinder Blood 93, 4059-4070 (1999)). Since the B cell response to CpGoligodeoxyribonucleotides (ODN) is mediated through TLR9 (Hemmi, et al.,Nature 408, 740-745 (2000)), we hypothesized that chromatin-containingIC could stimulate RF B cells by co-engaging TLR9. The TLR9-mediatedresponse to CpG S-ODN is distinguished from other known TLR signalingpathways by its presumed requirement for endosome acidification and/ormaturation as determined by sensitivity to chloroquine and ammoniumchloride (Yi, et al., J. Immunol. 160, 4755-4761 (1998); Hacker, et al.,EMBO J. 17, 6230-6240 (1998)). Concanamycin B and bafilomycin A arespecific inhibitors of the V-type ATPase responsible for acidificationof endosomes (Benaroch, et al., EMBO J. 14, 37-49 (1995)). To evaluatethe role of TLR9 in the activation of RF+ B cells, the effect ofchloroquine, concanamycin B, bafilomycin A, and ammonium chloride on thestimulatory capacity of mAb PL2-3 and known TLR2 (lipopeptide, porin B)(Massari, et al., J. Immunol. 168:1533-1537, 2002), TLR4 (LPS), and TLR9(CpG S-ODN 1826) ligands was determined. As expected, chloroquineinhibited the CpG S-ODN response and these inhibitors had little effecton the response to the TLR2 and TLR4 ligands. Notably, all four agentsthat blocked chloroquine also inhibited the RF+ B cell response to mAbPL2-3 (FIGS. 5A and 5B). The link to chloroquine is intriguing, aschloroquine is an effective treatment for autoimmune diseases includingrheumatoid arthritis and SLE (Canadian Hydroxychloroquine Study Group,N. Engl. J. Med. 324, 150-154 (1991); Furst, et al., Arth. Rheu. 42,357-365 (1999)). Our data therefore suggest that the therapeutic effectsof chloroquine are due, at least in part, to its ability to interferewith TLR-mediated signals that contribute to autoantibody production orthe production of proinflammatory mediators.

The B cell response to the stimulatory CpG S-ODN 1826 can also beblocked by a group of closely related inhibitory CpG S-ODN such as S-ODN2088 (Lenart, et al., Antisense Nucleic Acid Drug Dev. 4, 247-256(2001)). We found that S-ODN 2088 profoundly inhibited the proliferativeresponse to S-ODN 1826 but had no effect on the response to anti-IgM orour TLR2 and TLR4 ligands (FIGS. 5C and 5B). Most significantly, S-ODN2088 also dramatically blocked the RF+ B cell response to mAb PL2-3.Overall, these data strongly implicate endosomal processing and/orengagement of an endosome-associated TLR, most probably TLR9, in RF+ Bcell activation.

It has been clearly shown that autoreactive B cell clones can undergoisotype switching and somatic mutation, similar to the T-dependentfeatures of conventional B cell responses to foreign antigens(Shlomchik, et al., Nature 328, 805-811 (1987)). However, more recentreports have suggested that autoreactive B cells may segregate fromconventional B cells in the peripheral lymphoid tissues; autoreactive Bcells tend to localize in the marginal zone of the splenic white pulp(Zeng, et al., J. Immunol. 164, 5000-5004 (2000)), while conventional Bcells home to the follicular regions. In addition, in some instances,expansion and somatic mutation of autoreactive B cells can take placeoutside of the germinal center. These discrete localization patterns mayreflect the preferential accumulation of stimulatoryautoantibody/autoantigen IC at these sites. Alternatively, therelatively unique homing pattern may result from distinct chemokinereceptor profiles elicited in response to the combined effects ofBCR/TLR engagement. Future studies will need to compare the functionalproperties of B cells activated through BCR crosslinking alone from Bcells activated through BCR/TLR dual engagement.

Autoantibodies that recognize DNA/nucleosomes are the defining and mostprevalent specificity in SLE patients and murine lupus models of SLE,while RFs are characteristic of a subset of SLE patients,autoimmune-prone lpr mice, and RA patients. Remarkably, these sameautoantigens are the targets in a number of new murine autoimmunitymodels generated by mutations that disrupt lymphocyte homeostasis orautoantigen metabolism (Botto, et al., Nature Genetics 19, 56-59 (1998);Bickerstaff, et al., Nature Medicine 5, 694-697 (1999); Napirei, et al.,Nat. Gen. 25, 177-180 (2000); Scott, et al., Nature 411, 207-211(2001)). The reason for the predominance of these particularspecificities has been long-sought. Our data provide an explanation,namely the potent synergistic interaction of BCR/TLR signaling eventsmediated by chromatin containing immune complexes. In our experimentalsystem, dependent on endosome/lysosome-localized TLR9 (Hacker, et al.,EMBO J. 17, 6230-6240 (1998)), it is reasonable to conclude thatautoantibody/autoantigen IC engagement of the BCR triggers theendocytosis of IC-associated antigen that then results in the highlyefficient delivery of chromatin fragments to endosome-associated TLR9.In contrast to published studies (Bell, et al., J. Clin. Invest. 85,1487-1496 (1990); Bell, et al., Clin. Immunol. Immunopath. 60, 1326(1991)), we have been unable to activate B cells with either culturesupernatants or chromatin fragments alone. Whether other strains of micewill prove more responsive to our fragments remains to be determined.

Beyond the current experimental model, the principle of BCR/TLRdual-engagement has wide ranging implications for autoimmunity ingeneral. Model ligands such as haptenated-LPS and haptenatedLPSLPS-coupled SRBC can also co-signal BCRs and TLRs and are remarkablypotent and specific for the B cells that have the relevant receptors(Pike, Methods Enzymol. 150:265-75, 1987). Activation in this mode istherefore likely to be a fundamental event in the loss of peripheral Bcell tolerance in a wide variety of settings; other autoantigens maysignal through TLRs other than TLR9. Overall, the data establish acritical role for endogenous TLR ligands in the aberrant activation ofthe adaptive immune system in autoimmunity and explain why theautoantibody repertoire is often skewed toward the recognition ofsubcellular nucleic acid/protein particles (Tan, E. Adv. Immunol. 44,93-151 (1989)).

Methods

Mice. The MRL+/+AM14 BCR Tg mice described previously (Hannum, et al.,J. Exp. Med. 184, 1269-1278 (1996); Rifkin, et al., J. Immunol. 165,1626-1633 (2000)) were crossed to Cr2-deficient mice, kindly provided byDr. Michael Carroll (Harvard Medical School, Boston), and the F1offspring were intercrossed to generate AM14 Cr2 deficient and Cr2sufficient control mice. MyD88−/− mice, originally produced by Dr.Shizuo Akira (Osaka University, Osaka, Japan) (Adachi, et al., Immunity9, 143-150 (1998)) and kindly provided through Dr. Douglas Golenbock(University of Massachusetts Medical School, Worcester, Mass.) werecrossed to AM14 MRL+/+ mice to generate AM14 MyD88−/− and controllittermate offspring. The RF+ offspring were initially identified byPCR, and their identity was confirmed by flow cytometric analysis ofperipheral blood lymphocytes or spleen cells, using the 4-G7 monoclonalanti-idiotype as described (Shlomchik, et al., Int. Immunol. 5,1329-1341 (1993)). Complement receptor genotype was determined by flowcytometry using the FITC-conjugated 7G6 antibody (Pharmingen, San Diego,Calif.). MyD88 genotype was determined by PCR using the primers: MyD88F(5′-TGG CAT GCC TCC ATC ATA GTT AAC C-3′) (SEQ ID NO: 1), MyD88R (5′-GTCAGA AAC AAC CAC CAC CAT GC-3′) (SEQ ID NO: 2), and neoR (5′-ATC GCC TTCTAT CGC CTT CTT GAC G-3′) (SEQ ID NO: 3) (MWG Biotech, High Point, N.C.)to yield wild type and knockout products of approximately 550 bp and 750bp respectively.

Cell culture and reagents. Spleen cell preparations were T-depleted andcultured with the appropriate ligands for 40-48 hrs. In someexperiments, B cells were preincubated with CD40L as described (Rifkin,et al., J. Immunol. 165, 1626-1633 (2000)). Proliferation was assessedby ³[H]-thymidine incorporation during the final 16 hours of culture.Data are presented as the mean percentage of the anti-IgM response fromtriplicate cultures. The percentage of the anti-IgM response wascalculated according to the formula: [(cpm experimental condition−cpmCD40L alone)/(cpm anti-IgM−cpm CD40L alone)×100].

Ligands included: goat anti-mouse IgM F(ab′)₂ (15 μg/ml, JacksonImmunoResearch Laboratories, West Grove, Pa.); the nucleosome specificmAbs PR1-3 (IgG2a^(j)), PL2-3 (IgG2a^(j)), and PL2-8 (IgG2b) (all at 50μg/ml), kindly provided by Dr. Marc Monestier (Temple University Schoolof Medicine, Philadelphia, Pa.) (Monestier and Novick Mol. Immunol. 33,89-99 (1996)); 10 μg/ml LPS (Sigma, St. Louis, Mo.); 0.3-2 μg/mlstimulatory CpG S-ODN 1826 (Yi, et al., J. Immunol. 160, 4755-4761(1998)) (Oligo's Etc, Wilsonville, Ohio), 10 μg/ml Neisseriameningitidis porin B (kindly provided by Dr. Lee Wetzler, BostonUniversity School of Medicine, Boston, Mass.); 10 μg/ml syntheticlipopeptide Pam₃Cys-Sk₄ (Dr. G. Jung, Univ. of Tuebingen, Germany,kindly provided by Dr. Doug Golenbock); and the anti-TNP mAbs Hy1.2(IgG2a^(a)) and C1040 (IgG2a^(b)) (Hannum, et al., J. Exp. Med. 184,1269-1278 (1996)) (both at 50 μg/ml) complexed to varying concentrationsof TNP-BSA. In some experiments, DNase I (type IV) (Sigma, St. Louis,Mo.), chloroquine (Sigma), concanamycin B, bafilomycin A (Sigma), or 12μg/ml of the inhibitory CpG S-ODN 2008 (Lenart, et al., AntisenseNucleic Acid Drug Dev. 4, 247-256 (2001)) (Oligo's Etc.) were added tothe cultures 15-30 min-2 hr before the addition of the ligands. All mAband ODN preparations were shown to be endotoxin free by LimulusAmebocyte Lysate ELISA (Bio-Whittaker, Walkersville, Md.).

EXAMPLE 3

We have further shown that dendritic cells are activated bychromatin-containing immune complexes through sequential engagement ofthe Fc-receptor and TLR 9. This is a similar mechanism to that we havedescribed for B cells except that in the case of the B cell, therelevant cell surface receptor is the B cell antigen receptor, whereasin dendritic cells the relevant cell surface receptor is an Fc gammareceptor (FcγR). Both cell surface receptors serve to deliver chromatinto TLR9 located within the endosome/lysosome compartment.

In FIGS. 8A-8C we show that dendritic cells can be specificallyactivated through TLR9 and this activation can be blocked by TLR9inhibition with ODN 2088. These experiments demonstrate that thedendritic cells that we use in the subsequent experiments (shown inTables 1 and 2 below) express functional TLR9, in that the specific TLR9activating ligand ODN 1826 induces TNF-α and IL-12 production by thedendritic cells (8A, 8B) and furthermore induces upregulation ofco-stimulatory molecules (8C). ODN 2088 is a specific inhibitor of theTLR9 mediated activation because (as shown in 8A, 8B, 8C), ODN 2088blocks ODN 1826 mediated activation but has no effect on LPS mediatedactivation.

Dendritic cells were generated from the bone marrow of C57BL/6 mice byin vitro culture with GM-CSF and IL-4 for 6 days. On day 6, the CD11cpositive dendritic cells were isolated using magnetic beads (MiltenyiBiotec) and either pre-incubated, or not pre-incubated, for 30 minuteswith ODN 2088, an inhibitory oligodeoxynucleotide that specificallyblocks signaling through TLR9. The stimulatory ODN 1826 (CpG, 3 μg/ml),that specifically activates through TLR9, or lipopolysaccharide (LPS, 10μg/ml), that activates through TLR4, or culture medium only (media), wasthen added to the cultures. After 48 hours, levels of TNF-α (8A) andIL-12 (8B) in the culture supernatants were measured by ELISA (resultsshown as OD units determined by absorption at 405 nm). After removal ofthe supernatants, cells were collected and analyzed by flow cytometryfor expression of the co-stimulatory molecule CD86 (8 c). Unstimulatedrefers to the level of expression of CD86 in the presence of mediaalone, whereas stimulated refers to the level of expression of CD86 inthe presence of the stimulus (ODN 1826 or LPS).

Table 1 below shows that chromatin-containing immune complexes(containing autoantibody in a complex with the chromatin autoantigen)activate dendritic cells to produce TNF-α whereas immune complexescontaining a foreign antigen (TNP-BSA) do not induce this dendritic cellactivation. Additionally, the activation induced by thechromatin-containing immune complexes is completely blocked by the TLR9specific inhibitor ODN 2088, indicating that engagement of TLR9(presumably by the chromatin within the chromatin-containing immunecomplex) is an essential component of the activation process.

TABLE 1 Stimulus None 2088 Media (no stimulus) <50 <50 ODN 1826 2347 <50LPS 2514 2393 PL2-3 1653 <50 TNP/αTNP-BSA IC 1:1 <50 <50 TNP/αTNP-BSA IC4:1 <50 <50 TNP/αTNP-BSA IC 16:1 <50 <50

Table 1 shows that dendritic cell TNF-α production induced bychromatin-containing immune complexes is blocked by the TLR9 specificinhibitor ODN 2088. Dendritic cells were generated from the bone marrowof C57BL/6 mice by in vitro culture with GM-CSF and IL-4 for 6 days. Onday 6, the CD11c positive dendritic cells were isolated using magneticbeads (Miltenyi Biotec). The dendritic cells were then eitherpre-incubated, or not pre-incubated, for 30 minutes with ODN 2088 at 12μg/ml. The stimulatory ODN 1826 (3 μg/ml), that specifically activatesthrough TLR9, lipopolysaccharide (LPS, 10 μg/ml), that activates throughTLR4, the anti-chromatin antibody PL2-3 (50 μg/ml), three differentantibody/protein ratios of α-TNP/TNP-BSA immune complexes (50 μg/ml), orthe culture medium only (media), was then added to the cultures. After48 hours, levels of TNF-α in the culture supernatants was measured byELISA (results shown as pg/ml; lower level of sensitivity of assay is 50pg/ml). A representative experiment of 3 similar experiments is shown.

Table 2 shows that chromatin-containing immune complexes induce TNF-αproduction by dendritic cells from wildtype mice (as was also seen inthe studies shown in table 1 in the absence of ODN 2088) but completelyfail to induce TNF-α production by dendritic cells from Fc receptor γchain deficient mice. This demonstrates that an activating Fc receptoris absolutely required for dendritic cell activation bychromatin-containing immune complexes.

TABLE 2 Fc receptor γ Stimulus wildtype chain deficient Media (nostimulus) <50 <50 ODN 1826 7899 2373 LPS 7056 3411 PL2-3 4554 <50 PL2-83964 <50 TNP/αTNP-BSA IC 1:1 <50 <50 TNP/αTNP-BSA IC 4:1 <50 <50

Table 2 demonstrates that chromatin-containing immune complexes induceTNF-α production by dendritic cells from wildtype mice but do not induceTNF-α production by dendritic cells from Fc receptor γ chain deficientmice. Dendritic cells were generated from the bone marrow of C57BL/6wildtype mice and from C57BL/6 mice lacking the Fc receptor γ chain inby in vitro culture of the bone marrow cells with GM-CSF and IL-4 for 6days. On day 6, the CD11c positive dendritic cells were isolated usingmagnetic beads. The stimulatory ODN 1826 (3 μg/ml), that specificallyactivates through TLR9, lipopolysaccharide (LPS, 10 μg/ml), theanti-chromatin antibodies PL2-3 (50 μg/ml; IgG2a) and PL2-8 (50 μg/ml;IgG2b), two different antibody/protein ratios of α-TNP/TNP-BSA immunecomplexes (50 μg/ml), or the culture medium only (media), was then addedto the dendritic cell cultures. After 48 hours, levels of TNF-α in theculture supernatants was measured by ELISA (results shown as pg/ml;lower level of sensitivity of assay is 50 pg/ml). A representativeexperiment of 2 similar experiments is shown.

Taken together, the data shown in Table 1 and Table 2 above demonstratethat dendritic cell activation induced by chromatin-containing immunecomplexes requires a signal through an activating Fcγ receptor as wellas a signal mediated through TLR9.

Additional experiments to evaluate mechanisms and consequences of thisFcγR/TLR dual engagement pathway are outlined below.

Characterization of the IFN-α Producing Dendritic Cell Subsets.

Mouse strains. Differences in dendritic cell and macrophage functionbetween autoimmune lupus-like and non-autoimmune mouse strains have beendescribed. These include differences in immune complex handling (Joneset al., Clin. Immunol. Immunopathol. 36: 30-39, 1985; Magilavy et al.,J. Immunol. 131: 2784-2788, 1983), phagocytosis (Russell and SteinbergClin. Immunol. Immunopathol. 27: 387-402, 1983), Fc receptor expressionand function (Pritchard et al., Current Biology. 10: 227-230, 2000;Jiang et al., Immunogenetics. 51: 429-435., 2000) and cytokineproduction (Alleva et al., J. Immunol. 159: 5610-5619., 1997; Koh etal., J. Immunol. 165: 4190-4201., 2000). It is therefore important todirectly compare dendritic cells derived from both autoimmune andnon-autoimmune strains. MRL+/+ and (NZBxNZW) F1 are the two autoimmunelupus-like strains and C57BL/6 and BALB/c are the two non-autoimmunestrains that are used.

Purification of dendritic cell subsets. There are 2 dendritic cellsubsets which are particularly important in the pathogenesis of SLE:CD11c+ CD8α+ and CD11c+ B220+ Gr-1+. These cells are either obtaineddirectly from the spleen (Hochrein et al., J. Immunol. 166: 5448-5455,2000; Nakano et al., J. Exp. Med. 194: 1171-1178, 2001) or generated byin vitro culture of bone marrow cells in the presence of Flt3 ligand(Brasel et al., Blood. 96: 3029-3039, 2000; Gilliet et al., J. Exp. Med.195: 953-958, 2002). Splenic dendritic cells are isolated as described(Vremec et al., J. Immunol. 164: 2978-2986, 2000) and, after appropriatefluorescent antibody staining, the specific dendritic cell subsets arefractionated and collected using a MoFlo cell sorter (Cytomation, FortCollins, Colo.). The CD11c+ CD8α− dendritic cell subset, which is alsofound in the spleen, are isolated at the same time as the target CD11c+CD8α+ dendritic cell subset and used as an experimental control.

To generate bone marrow derived dendritic cells, bone marrow cells areharvested and cultured in vitro for 6-10 days in the presence ofrecombinant murine Flt3 ligand using established protocols (Gilliet etal., J. Exp. Med. 195: 953-958, 2002; Labeur et al., J. Immunol. 162:168-175, 1999). Bone marrow cells grown in Flt3 ligand are enriched inthe target populations, namely CD11c+ CD8+ and CD11c+ B220+ Gr-1+dendritic cells. These populations are fractionated by cell sorting asdescribed above for the spleen-derived dendritic cells.

Determining the expression of TLR9 in the specific dendritic cellsubsets. TLR9 messenger RNA is measured by quantitative reversetranscription polymerase chain reaction (RT-PCR). In addition, theresponse of the dendritic cell subsets to a TLR9-specific stimulatoryligand, S-ODN 1826 (Ballas et al., J. Immunol. 167: 4878-4886, 2001),are compared by measuring expression of co-stimulatory molecules andcytokine production. Specific antibodies against murine TLR9 can beproduced using techniques known to one skilled in the art.

Preparation of IC and Assessment of Ic Binding and Uptake by DendriticCells.

Immune complex preparation. Anti-nucleosome/chromatin IC is prepared byincubating the anti-nucleosome monoclonal antibodies PL2-3 (IgG2a),PL2-8 (IgG2b) and PL9-7 (IgG3) with supernatant obtained from 24 hour invitro cultures of spleen cells. Chromatin is spontaneously released fromspleen cells in culture (Bell et al., J. Clin. Invest. 85: 1487-1496,1990) and binding of the anti-nucleosome antibodies to this chromatinresults in the formation of IC which can be measured using a Clqimmunoassay (Rifkin et al., Journal of Immunology. 165: 1626-1633,2000). We have further shown that biotinylated PL2-3 preincubated withculture fluid binds specifically to rheumatoid factor B cells asdetected by flow cytometry; monomeric biotinylated PL2-3 does not bind.Non-self antigen control IC is made using the anti-TNP monoclonalantibodies Hy1.2 (IgG2a) or C4010 (IgG2b) and incubating them withTNP-BSA, with confirmation of IC formation also by Clq binding asdemonstrated (Leadbetter et al., Nature. 416: 603-607, 2002). Additionalnon-self antigen control IC is made using the anti-ovalbumin monoclonalantibodies Ov1 (IgG2a) and Ov2 (IgG2b) prepared in this laboratory andincubating them with ovalbumin. As well as demonstrating IC formation ina Clq binding assay, FPLC is also used as an additional confirmatoryassay.

Immune complex binding and uptake by dendritic cells. Before setting upthe actual experimental cultures containing the IC and dendritic cells,it is necessary to determine the extent to which the purified dendriticcell subsets from the different strains are able to bind and take up IC.This is particularly important given the reported differences betweenautoimmune and non-autoimmune mouse strains as regards IC handling(Jones et al., Clin. Immunol. Immunopathol. 36: 30-39, 1985; Magilavy etal., J. Immunol. 131: 2784-2788, 1983) and Fc receptor expression(Pritchard et al., Current Biology. 10: 227-230, 2000; Jiang et al.,Immunogenetics. 51: 429-435, 2000). The experimental approach is totrack the antibody component of the immune complex using a combinationof flow cytometry and confocal microscopy. The anti-nucleosomeantibodies PL2-3 (IgG2a), PL2-8 (IgG2b) and PL9-7 (IgG3) arebiotin-conjugated, using standard procedures, andbiotin-anti-nucleosome/chromatin IC are prepared as above. To confirmthat the biotin-anti-nucleosome/chromatin IC retains functionality it isassessed whether the biotin-PL2-3/chromatin IC is able to induceproliferation in rheumatoid factor B cells to the same extent as thenon-biotin-PL2-3/chromatin IC (Rifkin et al., Journal of Immunology.165: 1626-1633, 2000). The biotin-PL2-8/chromatin IC and thebiotin-PL9-7/chromatin IC cannot be tested in this way because IgG2b andIgG3 are not recognized by the rheumatoid factor B cell receptor, but itis assumed that the functional effect of biotin-conjugation will besimilar irrespective of isotype. Isotype-matched biotin-anti-TNP/TNP-BSAIC and biotin-anti-ovalbumin/ovalbumin IC are prepared similarly.Biotin-conjugated antibodies alone not made into complexes are used ascontrols. The biotin-conjugated IC and biotin-conjugated antibodiesalone are added to the different dendritic cell subsets, and flowcytometry is used to detect cell surface binding. The biotin-labeledcompounds are visualized with an anti-biotin monoclonal antibodyconjugated to a specific fluorochrome (Molecular Probes: anti-biotinmouse monoclonal 2F5 Alexa Fluor 488 conjugate). Subsequently, confocalmicroscopy is used to assess dendritic cell internalization of theanti-nucleosome antibody (biotin-anti-nucleosome/chromatin IC), theanti-TNP antibody (biotin-anti-TNP/TNP-BSA IC) or the anti-ovalbuminantibody (biotin-anti-ovalbumin/ovalbumin IC) following incubationperiods of 1-12 hours and fixation with 3% paraformaldehyde.

In addition, co-localization studies are performed by co-staining withfluorescent-labeled monoclonal antibodies specific forendosomal/lysosomal compartments, or by the use of fluorescent pHindicators that partition into acidic organelles (Molecular Probes).

Measurement of Dendritic Cell Activation by Chromatin-Containing IC andEstablishment of the Role of Toll-Like Receptors and Fc Gamma Receptors.

Measuring dendritic cell activation by IC. The anti-nucleosome/chromatinIC, anti-TNP/TNP-BSA IC and anti-ovalbumin/ovalbumin IC outlined aboveare incubated with the dendritic cell subsets and dendritic cellactivation determined by measuring upregulation of co-stimulatorymolecules and cytokine production. Controls include the monomericnon-complexed antibodies alone, antigens alone, stimulatory CpG S-ODN1826 as a positive control for TLR9 signaling, LPS as a positive controlfor TLR4 signaling and porin B as a positive control for TLR2 signaling.Cytokines are measured by ELISA and include IL-12, TNF-α, IL-10 andIFN-α. IL-12 and TNF-α are the two cytokines most commonly used asmarkers of dendritic cell activation (Banchereau et al., Annu. Rev.Immunol. 18: 767-811, 2000) and IFN-α is the cytokine most stronglyassociated with the activation of the CD11c+ CD8α+ and the CD11c+ B220+Gr-1+ dendritic cell subsets. It is necessary to measure IL-10 as it hasbeen shown that Fc receptor engagement in macrophages can lead to IL-10production induced by a subsequent stimulus which, in the absence of Fcreceptor engagement, leads to IL-12 and not IL-10 production (Gerber andMosser J. Immunol. 166: 6861-6868., 2001). The increased expression ofMHC class II and the co-stimulatory molecules CD80, CD86, and CD40 isdetermined by flow cytometry.

Role of Toll-like receptors and Fc gamma receptors in IC-mediateddendritic cell activation. S-ODN 2088 is an inhibitory CpG S-ODN whichspecifically blocks signaling through TLR9 (Lenert et al., Antisense andNucleic Acid Drug Development. 11: 247-256, 2001). We have shown thatS-ODN 2088 strongly inhibits the chromatin-containing IC-inducedproliferation of B cells from rheumatoid factor B cell receptortransgenic mice (Leadbetter et al., Nature. 416: 603-607, 2002). Also,we have shown that S-ODN 2088 completely prevents dendritic cellactivation by stimulatory CpG S-ODN (such as S-ODN 1826) acting throughTLR9. The experiments outlined above are done in the presence or absenceof S-ODN 2088 to assess the role of TLR9 in IC-mediated dendritic cellactivation. They are also done in the presence and absence of inhibitorsof endosomal acidification, including chloroquine, concanamycin B andbafilomycin A, which prevent signaling through TLR9. In addition,dendritic cells from C57BL/6 MyD88+/+ mice are compared to dendriticcells from C57BL/6 MyD88−/− mice to establish the requirement for TLRsignaling.

In order to assess the requirement for Fc gamma receptor (FcγR)signaling, experiments are also done in the presence and absence of2.4.G2, a monoclonal antibody that specifically blocks murine FcγRII andFcγRIII (Araujo-Jorge et al., Infect Immun. 61: 4925-4928, 1993).However, the third type of murine Fc gamma receptor, FcγR I, is notblocked by 2.4G2 and can mediate IC-induced inflammatory responses(Ioan-Facsinay et al., Immunity. 16: 391-402., 2002; Barnes et al.,Immunity. 16: 379-389, 2002). Dendritic cells express all three classesof FcγR (FcγRI, FcγRII and FcγRIII) (Ravetch and Bolland, Ann. Rev.Immunol. 19: 275-290, 2001). Therefore, additional studies can beperformed using FcγR knockout mice. The mice are available from JacksonLaboratories, and include i) mice rendered genetically deficient inFcγRI and FcγRIII by knockout of the common stimulatorysignal-transducing gamma chain shared by these two receptors (Takai etal., Cell. 76: 519-529., 1994) ii) FcγRII knockout mice (Takai et al.,Nature. 379: 346-349., 1996) and iii) FcγRIII knockout mice (Hazenbos etal., Immunity. 5: 181-188., 1996).

Role of IC and TLR9 in antigen processing and presentation by dendriticcells. CD4+ autoreactive T cells are central to the pathogenesis of SLE,both in humans and in murine models of the disease (Craft et al.,Immunol. Res. 19, 1999; Hoffman, Front. Biosci. 6: D1369-78, 2001;Shlomchik et al., Nature Rev. Immunol. 1: 147-153., 2001). Dendriticcells are the key antigen presenting cells involved in initiating T cellresponses (Banchereau et al., Annu. Rev. Immunol. 18: 767-811, 2000) andTLR9 engagement has been shown to strongly promote development ofTh1-type immune responses (Jakob et al., J. Immunol. 161: 3042-3049,1998; Lipford et al., J. Immunol. 165: 1228-1235, 2000). To show whetherTLR9 engagement alters the T cell response to antigen contained withinimmune complexes, ovalbumin is conjugated to the stimulatory CpG S-ODN1826 as described (Shirota et al., J. Immunol. 164: 5575-5582., 2000).IC is consequently made by incubating unconjugated ovalbumin or theovalbumin-CpG conjugate with the anti-ovalbumin monoclonal antibodiesOv1 (IgG2a) or Ov2 (IgG2b). Dendritic cell subsets from BALB/c mice(H-2^(d)) are pulsed with these IC. CD4+ T cells, which recognizeovalbumin in the context of H-2A^(d), are purified from the lymph nodesof ovalbumin-specific T cell receptor transgenic mice (DO11.10 mice)(Murphy et al., Science. 250: 1720-1723, 1990). These CD4+ T cells arecultured with the IC-pulsed dendritic cells and T cell proliferation andcytokine production (IFN-gamma, IL-4 and IL-2) is measured duringprimary and secondary T cell responses. S-ODN 2088 and endosomalacidification inhibitors is used to assess the role of TLR9.

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The references cited herein and throughout the specification are hereinincorporated by reference in their entirety.

1. A method of treating a patient having or at risk of having an ImmuneComplex Associated Disease (ICAD) or a systemic autoimmune disease,comprising administering to a patient having or at risk of having anICAD or a systemic autoimmune disease an effective amount of a compoundthat inhibits immune complexes or autoantigens from binding to oractivating a Toll-like receptor (TLR), wherein the Toll-like receptor ischosen from Toll-like receptor 9 (TLR9) and Toll-like receptor 3 (TLR3)and wherein said immune complex comprises an autoantibody and anautoantigen bound to a cell receptor, to treat the ICAD or systemicautoimmune disease.
 2. The method of claim 1, wherein the ICAD issystemic lupus erythematosus.
 3. The method of claim 1, wherein the ICADis rheumatoid arthritis.
 4. The method of claim 1, wherein the ICAD ishepatitis-C related immune complex disease.
 5. The method of claim 1wherein the Toll-like receptor is TLR9.
 6. The method of claim 1 whereinthe Toll-like receptor is TLR3.
 7. The method of claim 1, wherein thecompound is selected from the group consisting of (a) compounds thatbind components of the immune complex and prevent its formation orbinding to the Toll-like receptor; (b) Toll-like receptor decoys; (c)compounds that inhibit the activity of MyD88 or other components of aTLR-initiated signaling cascade; (d) compounds that inhibit productionof immune complex components; and (e) Toll-like receptor antagonists. 8.The method of claim 7, wherein the Toll-like receptor antagonist is aninhibitory oligonucleotide.
 9. The method of claim 7, wherein theToll-like receptor antagonist is a dominant negative Toll-like receptor.10. The method of claim 1, wherein the compound is an antibody thatbinds a Toll-like receptor.
 11. The method of claim 10, wherein theantibody is a single chain antibody.
 12. The method of claim 1, whereinthe compound comprises a cocktail of compounds that inhibit binding to acombination of at least two of Toll-like receptor 2 (TLR2), TLR3, andTLR9.
 13. A method for screening for compounds that inhibit immunecomplex formation or binding to a Toll-like receptor, comprisingcontacting immune complex components with a compound being screened andwith a TLR chosen from Toll-like receptor 3 (TLR3) and Toll-likereceptor 9 (TLR9); measuring binding of an antigenic fragment of animmune complex to the TLR, wherein said immune complex comprises anautoantibody and an autoantigen; and identifying the compound beingscreened as an inhibitor of immune complex formation or of binding to aToll-like receptor when the binding with the compound is reducedcompared to binding without the compound.
 14. The method of claim 13,wherein the TLR is TLR9.
 15. The method of claim 13, wherein the bindingwith the compound is reduced at least 50 percent compared to bindingwithout the compound.
 16. A method for screening for compounds thatinhibit immune complex formation or binding to a Toll-like receptor,comprising contacting immune complex components with a compound beingscreened and with a dendritic cell that expresses a TLR chosen fromToll-like receptor 3 (TLR3) and Toll-like receptor 9 (TLR9); measuringactivation of the dendritic cell; and identifying the compound beingscreened as an inhibitor of immune complex formation or of binding to aToll-like receptor when activation of the dendritic cell with thecompound is reduced compared to activation of the dendritic cell withoutthe compound.
 17. The method of claim 16, wherein the TLR is TLR9. 18.The method of claim 16, wherein the measuring activation of thedendritic cell comprises measuring production of a cytokine chosen fromTNF-α, interferon-α, and BAFF.
 19. The method of claim 16, wherein themeasuring activation of the dendritic cell comprises measuringupregulated expression on the dendritic cell of a costimulatory moleculechosen from CD80, CD86, and MHC class II.
 20. The method of claim 16,wherein the activation of the dendritic cell with the compound isreduced at least 50 percent compared to activation of the dendritic cellwithout the compound.
 21. The method of claim 16, wherein the immunecomplex components comprise an autoantibody and an autoantigen.
 22. Amethod for screening for compounds that inhibit immune complex formationor binding to a Toll-like receptor, comprising contacting immune complexcomponents with a compound being screened and with a B cell thatexpresses a TLR chosen from Toll-like receptor 3 (TLR3) and Toll-likereceptor 9 (TLR9); measuring activation and/or proliferation of the Bcell; and identifying the compound being screened as an inhibitor ofimmune complex formation or of binding to a Toll-like receptor whenactivation and/or proliferation of the B cell with the compound isreduced compared to activation and/or proliferation of the B cellwithout the compound.
 23. The method of claim 22, wherein the TLR isTLR9.
 24. The method of claim 22, wherein the measuring activation ofthe B cell comprises measuring upregulated expression on the B cell of acostimulatory molecule chosen from CD80, CD86, and MHC class II.
 25. Themethod of claim 22, wherein the activation and/or proliferation of the Bcell with the compound is reduced at least 50 percent compared toactivation and/or proliferation of the B cell without the compound. 26.The method of claim 22, wherein the immune complex components comprisean autoantibody and an autoantigen.